Nucleotide Compositions Comprising Photocleavable Markers And Methods Of Preparation Thereof

ABSTRACT

Labelled nucleotides and polynucleotides useful in the sequencing of nucleic acids are described. Methods of preparing photocleavable marker nucleotides and photocleavable marker-polynucleotide conjugates are described. Such photocleavable marker nucleotides can be incorporated into nucleic acid so as to create photocleavable marker-polynucleotide conjugates.

FIELD OF THE INVENTION

The present invention generally relates to nucleotides andpolynucleotides useful in the sequencing of nucleic acids. The presentinvention specifically relates to compositions comprising nucleotidesand polynucleotides comprising photocleavable labels and the methods ofpreparing said compositions.

BACKGROUND OF THE INVENTION

The sequencing of nucleic acids is one the most powerful and valuabletools for scientific research. As evidenced by the Human Genome project,there is an ever increasing demand for nucleic acid sequenceinformation. There are numerous methods available for sequencing ofnucleic acids. The first methods were developed almost twenty years ago.For example, the Sanger enzymatic (i.e., dideoxy chain termination)method involves synthesis of a DNA strand from a single-strandedtemplate by a DNA polymerase. The Maxam and Gilbert method involveschemical degradation (i.e. chemical cleavage) of the original DNA. Bothmethods produce populations of radio-labelled polynucleotides that beginat a fixed point in the DNA to be sequenced and terminate at pointswhich are dependent upon the location of a particular base in theoriginal DNA strand. These polynucleotides are separated by apolyacrylamide gel electrophoresis, and the order of the nucleotides inthe original DNA is directly read from an autoradiograph of the gel.However, the time-consuming electrophoresis step associated with thesemethods is difficult to perform in a highly parallel (i.e. greater than1000 samples at a time per instrument) fashion.

Although both the Sanger and Maxam-Gilbert methods are currently used,there have been many changes and improvements. The enzymatic chaintermination method is probably the most popular and widely usedtechnique for sequence determination, especially since the automation ofthe procedure has been accomplished through use of fluorescent, ratherthan radioactive labelling, and the utilization of amplificationtechnology. The incorporation of amplification technology (e.g., thepolymerase chain reaction [PCR]) enables the sequencing reaction to becycled. Other advances include sequencing by chemiluminescence,multiplexing, and solid phase sequencing.

Other nucleic acid sequencing methods, such as sequencing byhybridization and pyrosequencing, have been developed that eliminate theelectrophoresis step associated with the Sanger and Maxam and Gilbertmethods, thereby allowing more samples to be sequenced in parallel.However, such methods often involve either lengthy cloning andamplification steps, or a time-consuming chemical cleavage step whereina fluorescently-labeled polynucleotide is removed by enzymaticdigestion.

Therefore, what is need is are compositions and methods that reduce thecomplexity of and time-consuming nature of parallel nucleic acidsequencing.

SUMMARY OF THE INVENTION

The present invention generally relates to nucleotides andpolynucleotides useful in the sequencing of nucleic acids. The presentinvention specifically relates to compositions comprising nucleotidesand polynucleotides comprising photocleavable labels and the methods ofpreparing said compositions.

The present invention contemplates compositions comprisingphotocleavable marker-polynucleotide conjugate compounds having thegeneral formula (I):

wherein X is selected from the group consisting of a phosphate group anda hydrogen atom, M is a photocleavable marker, B is a nucleobase,PC-linker is a photocleavable linker, and S is a sugar moiety.

It is not intended that the compounds of general formula (I) be limitedto a specific phosphate group. In one embodiment, said phosphate groupis a monophosphate group, more preferably a polyphosphate such as adiphosphate group, and even more preferably a triphosphate group. Inanother embodiment, said phosphate group is a pyrophosphate.

It is not intended that the nucleobase of the compounds of generalformula (I) be limited to a specific nucleobase. In one embodiment, saidnucleobase is selected from the group consisting of adenine, cytosine,guanine, thymine, uracil, and analogs thereof such as, for example,acyclic nucleobases.

It is not intended that the sugar moiety of the compounds of generalformula (I) be limited to a specific sugar moiety. In one embodiment,said sugar moiety selected from the group consisting of ribose,deoxyribose, dideoxyribose, and analogs thereof.

It is not intended that the photocleavable linker of the compounds ofgeneral formula (I) be limited to a specific photocleavable linker. Inone embodiment, said photocleavable linker is a photocleavable linkercomprising a protective group selected from the group consisting of9-fluorenylmethoxycarbonyl (Fmoc), 2-(4-biphenyl)propyl(2)oxycarbonyl(Bpoc), and derivatives thereof.

It is not intended that the compounds of general formula (I) be limitedto any specific photocleavable marker. In one embodiment, saidphotocleavable marker is BODIPY-FL. In another embodiment, saidphotocleavable marker is Cy5.

It is not intended that the photocleavable marker of the compounds ofgeneral formula (I) be detected by any specific method. In oneembodiment, said photocleavable marker is a binding member and isdetected via a second binding member. In another embodiment, saidphotocleavable marker is a molecule that can be detected by massspectrometry. In another embodiment, said photocleavable marker is afluorescent moiety and can be detected by fluorescence spectroscopy. Ina further embodiment, said photocleavable marker is a chelator capableof forming luminescent complexes.

The present invention also contemplates compositions comprisingphotocleavable marker-polynucleotide conjugate compounds having thegeneral formula (II):

wherein X is selected from the group consisting of a phosphate group anda hydrogen atom, M is a photocleavable marker, B is a nucleobase, and Sis a sugar moiety.

It is not intended that the compounds of general formula (II) be limitedto a specific phosphate group. In one embodiment, said phosphate groupis a monophosphate group, more preferably a polyphosphate such as adiphosphate group, and even more preferably a triphosphate group. Inanother embodiment, said phosphate group is a pyrophosphate.

It is not intended that the nucleobase of the compounds of generalformula (II) be limited to a specific nucleobase. In one embodiment,said nucleobase is selected from the group consisting of adenine,cytosine, guanine, thymine, uracil, and analogs thereof such as, forexample, acyclic nucleobases.

It is not intended that the sugar moiety of the compounds of generalformula (II) be limited to a specific sugar moiety. In one embodiment,said sugar moiety selected from the group consisting of ribose,deoxyribose, dideoxyribose, and analogs thereof.

It is not intended that the photocleavable linker of the compounds ofgeneral formula (II) be limited to a specific photocleavable linker. Inone embodiment, said photocleavable linker is a photocleavable linkercomprising a protective group selected from the group consisting of9-fluorenylmethoxycarbonyl (Fmoc), 2-(4-biphenyl)propyl(2)oxycarbonyl(Bpoc), and derivatives thereof.

It is not intended that the compounds of general formula (II) be limitedto any specific photocleavable marker. In one embodiment, saidphotocleavable marker is BODIPY-FL. In another embodiment, saidphotocleavable marker is Cy5.

It is not intended that the photocleavable marker of the compounds ofgeneral formula (II) be detected by any specific method. In oneembodiment, said photocleavable marker is a binding member and isdetected via a second binding member. In another embodiment, saidphotocleavable marker is a molecule that can be detected by massspectrometry. In another embodiment, said photocleavable marker is afluorescent moiety and can be detected by fluorescence spectroscopy. Ina further embodiment, said photocleavable marker is a chelator capableof forming luminescent complexes.

The present invention also relates to methods of preparingphotocleavable marker nucleotides. For example, in one embodiment, thepresent invention contemplates a method of preparing amarker-photocleavable linker-nucleotide conjugate (“photocleavablemarker nucleotide”) comprising: a) providing i) a photocleavable linkercomprising a protective group, ii) a nucleotide (or analog thereof), andiii) an activated marker molecule; b) operably linking saidphotocleavable linker to said nucleotide (or analog thereof) to producea photocleavable linker-nucleotide conjugate; c) removing saidprotective group from said photocleavable linker-nucleotide conjugateunder conditions such that an activated photocleavable linker-nucleotideconjugate is created, wherein said activated photocleavablelinker-nucleotide conjugate has an exposed reactive site on the linkerportion of the conjugate; and d) contacting said activated markermolecule with said activated photocleavable linker-nucleotide conjugateunder conditions such that a marker-photocleavable linker-nucleotideconjugate is produced. In a preferred embodiment, the method of thepresent invention produces a photocleavable marker nucleotide comprisinga nucleotide 5′-triphosphate.

Importantly, in a preferred embodiment, the conditions for step c) arechosen such that the integrity of said nucleotide is preserved. That isto say, the protective group is removed without removing substituents(e.g. functional groups) of the nucleotide.

It is not intended that the method of the present invention be limitedto a nucleotide having a particular phosphate group. In one embodiment,said nucleotide comprises a 5′-monophosphate group, more preferably a5′-diphosphate group, and even more preferably, a 5′-triphosphate group.The present invention also contemplates nucleotides having5′-polyphosphates consisting of more than three phosphate groups.

It is not intended that the method of the present invention be limitedto a specific activated marker molecule. In one embodiment, saidactivated marker molecule is BODIPY-FL-SE. In another embodiment, saidactivated marker molecule is Cy5-NHS.

The present invention also contemplates methods of preparingphotocleavable marker-polynucleotide conjugates. For example, in oneembodiment, the present invention contemplates a method of preparingphotocleavable marker-polynucleotide conjugates comprising: a) providingi) an unmodified polynucleic acid, ii) a photocleavable markernucleotide, and iii) a nucleic acid-modifying enzyme; b) contacting (ormixing or reacting or incubating) said polynucleic acid with saidphotocleavable marker nucleotide and said modifying enzyme underconditions such that said photocleavable marker nucleotide isincorporated into said polynucleic acid to produce a labeled polynucleicacid. In a preferred embodiment, the method further comprises: c)detecting said incorporated photocleavable marker (or incorporatedphotocleavable marker-nucleotide) in said labeled polynucleic acid. Inone embodiment, the method further comprises (prior to step c):separating unincorporated photocleavable marker nucleotide from saidlabeled polynucleic acid. Optionally, the method may further comprisethe step of removing the incorporated photocleavable marker from saidlabeled polynucleic acid by exposing said labeled polynucleic acid toelectromagnetic radiation, thereby creating treated polynucleic acid.

The present invention also contemplates the above method of preparing aphotocleavable marker-polynucleotide conjugate comprising an additionalstep of subjecting said treated polynucleic acid to a subsequentlabeling reaction with a different photocleavable marker nucleotideafter said removing step. The present invention also contemplates theabove method of preparing a photocleavable marker-polynucleotideconjugate wherein said contacting is performed in the presence of atemplate selected from the group consisting of polynucleic acid, DNA,RNA, cDNA, oligonucleotides.

It is not intended that the method of preparing a photocleavablemarker-polynucleotide conjugate of the present invention be limited toany specific nucleic acid-modifying enzyme. In one embodiment, saidnucleic acid-modifying enzyme is a DNA polymerase. In anotherembodiment, said nucleic acid-modifying enzyme is an RNA polymerase. Ina preferred embodiment, said nucleic acid-modifying enzyme is terminaldeoxynucleotidyl transferase.

It is not intended that the method of preparing a photocleavablemarker-polynucleotide conjugate of the present invention be limited to aparticular means by which said incorporated photocleavable markernucleotide on said polynucleic acid is detected. In one embodiment, saidincorporated photocleavable marker nucleotide on said polynucleic acidis detected by a means selected from the group consisting ofluminescence, fluorescence, chemiluminescence and mass spectrometry.

It is not intended that the method of preparing a photocleavablemarker-polynucleotide conjugate of the present invention be limited tothe use of a single photocleavable marker nucleotide. In one embodiment,a plurality of photocleavable marker nucleotides is provided, each ofsaid photocleavable marker nucleotides having a different markermolecule capable of being independently detected.

It is not intended that the method of preparing a photocleavablemarker-polynucleotide conjugate of the present invention be limited tothe use of a photocleavable marker nucleotide comprising a particularphosphate group. In one embodiment, said photocleavable markernucleotide is a nucleotide 5′-monophosphate, more preferably anucleotide 5′-diphosphate, and even more preferably, a nucleotide5′-triphosphate. The present invention also contemplates photocleavablemarker nucleotides having 5′-polyphosphates consisting of more thanthree phosphate groups.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts one example of the general structure of thephotocleavable marker-nucleotide conjugates of the present invention,compounds of general formula (I), wherein X is selected from the groupconsisting of a phosphate group and a hydrogen atom, M is aphotocleavable marker, B is a nucleobase, and S is a sugar moiety.

FIG. 2 shows one example of the incorporation of a photocleavablemarker-nucleotide conjugate into polynucleic acid, detection of themarker, and removal of the marker by photocleavage, subsequentincorporation of the same (or a different) photocleavablemarker-nucleotide conjugate into the same polynucleic acid and itssubsequent detection, followed by separation on denaturingpolyacrylamide gel and fluorescence imaging.

FIG. 3 depicts one example of a synthesis scheme for BODIPY-FL-PC-aadUTP(compound 6) and Cy5-PC-aadUTP (compound 7).

FIG. 4 depicts the chemical structures of BODIPY-FL-PC-aadUTP (compound6) and Cy5-PC-aadUTP (compound 7)

FIG. 5 shows the results of the high performance liquid chromatography(HPLC) starting material (compound 3), compound 4, compound 4 after UVillumination, and compound 5. Note that the retention time of compound 4(after illumination) regenerates the starting material as expected.

FIG. 6 shows the results of the HPLC of compounds 6 and 7 before andafter UV irradiation. Note that after UV illumination, compounds 6 and 7convert to starting material (compound 3) which shows successfulfluorophore removal upon UV exposure.

FIG. 7 shows the UV-VIS absorbance spectra of the starting material(compound 3, solid line) compared with that of compound 5 (dashed line).For compound 5, an increase in absorption at ˜270 nm was observed withabsorption extending towards ˜370 nm, as expected.

FIG. 8 shows the UV-VIS absorbance spectra of compound 6 (dashed line)compared with that of BODIPY-FL dye (solid line). A characteristic bandwith a maximum at ˜270 nm was observed for compound 6 in addition tofluorophore absorption with a maximum at ˜502 nm. This feature isconsistent with the presence of amidoallyluridine/2-nitrophenyl-ethylgroup.

FIG. 9 shows the UV-VIS absorbance spectra of compound 7 (dashed line)compared with that of Cy5 dye (solid line). A characteristic band with amaximum at ˜270 nm was observed for compound 7 in addition tofluorophore absorption with a maximum at ˜650 nm. This feature isconsistent with the presence of amidoallyluridine/2-nitrophenyl-ethylgroup.

FIG. 10 depicts one example of the incorporation of compound 6 into anoligonucleotide followed by: labeling with fluorescein-11-dUTP (lane 1);labeling with mixture of fluorescein-1-dUTP and compound 6(BODIPY-FL-PC-dUTP)(lane 2); labeling with BODIPY-FL-PC-dUTP (lane 3);BODIPY-FL-PC-dUTP only (i.e. no DNA)(lane 4); or fluorescein-11-dUTPonly (i.e. no DNA)(lane 5).

FIG. 11 depicts one example of the incorporation of compound 6 into anoligonucleotide and fluorescent marker removal after incorporationfollowed by separation on 7M urea/15% polyacrylamide gel and fluorescentimaging. Lane 1—labeling with BODIPY-FL-PC-dUTP; lane 2—labeling withBODIPY-FL-PC-dUTP followed by UV light irradiation of the reactionmixture prior to gel analysis.

FIG. 12 depicts one example of the compounds of general formula (II)wherein X is selected from the group consisting of a phosphate group anda hydrogen atom, M is a photocleavable marker, B is a nucleobase, and Sis a sugar moiety.

FIG. 13 depicts a further example of the compounds of general formula(II), as described in FIG. 12, wherein the sugar moiety is adeoxyribose.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are described.For purposes of the present invention, the following terms are definedbelow.

As used herein, the term “marker” refers to any atom or molecule whichcan be used to provide a detectable (preferably quantifiable) signal,and which can be attached to a nucleotides, polynucleotides, or nucleicacids (including polynucleic acids). Markers may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, and thelike. Such markers can be added to the nucleotides and polynucleotidesof the present invention. Marker molecules are “capable of beingindependently detected” where, in a mixture comprising two or moredifferent markers, each marker has a separate and distinct detectable(preferably quantifiable) signal. For example, the present inventioncontemplates a photocleavable marker-polynucleotide conjugatescomprising a plurality of different photocleavable marker moleculeswherein each molecule emits a distinct signal only at a specificwavelength of UV light.

Various methods of adding markers to nucleotides, polynucleotides, ornucleic acids are known in the art and may be used. Examples of markersfor nucleotides, polynucleotides, or nucleic acids include, but are notlimited to, the following: radioisotopes (e.g. ³H), fluorescent markers(e.g. BODIPY, Cy5, Cy3, FITC, rhodamine, and lanthanide phosphors),enzymatic markers (e.g. horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), biotinyl groups, pre-determinedpolypeptide epitopes recognized by a secondary reporter (e.g. leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, and epitope tags). In some embodiments, markers areattached by linkers, or spacer arms, of various lengths to reducepotential steric hindrance.

As used herein, the term “photocleavable marker” refers to a marker thatmay be removed from a nucleotide, polynucleotide, chemical group, ornucleic acid, to which it is attached or operably linked, by exposure toelectromagnetic radiation (e.g. visible light, UV light, etc.). Thewavelength of light necessary to photocleave the marker is dependentupon the structure of the photocleavable marker used. The presentinvention contemplates compositions comprising photocleavable markersthat are chemical compounds containing a 2-nitrobenzyl moiety such as,for example, compounds 6 & 7 as depicted in FIG. 3, andN-hydroxysuccinimidyl-4-azidosalicyclic acid (NHS-ASA). The terms“photocleavable marker-nucleotide” and “photocleavable marker-nucleotideconjugate” refer to compounds comprising a photocleavable marker that isoperably linked to a nucleotide or polynucleotide group. The term“plurality of photocleavable marker nucleotides” as used hereindesignates that more than one such marker nucleotide is utilized,wherein said plurality comprises two or more different photocleavablemarker nucleotides.

As used herein, the term “chelator” refers to a ligand that contains twoor more atoms, each of which can simultaneously form a two-electrondonor bond (i.e. chelate) to the same metal ion. A “chelator” may alsobe referred to as a polydentate ligand.

As used herein, the phrase “the photocleavable marker is a chelatorcapable of forming luminescent complexes,” refers to a photocleavablemarker molecule comprising a portion that chelates a metal ion (e.g.Terbium, Europium, Samarium, Ruthenium, Calcium, Magnesium, Manganese,Iron, Copper, Cobalt, Nickel, or other polyvalent cations) wherein thechelating of said metal ion allows detection by luminescence. Forexample, the present invention contemplates a photocleavable marker thatis a first chelator (e.g. salicylic acid) capable of forming luminescentcomplex when reacted with a second chelator (e.g. EDTA) and a metal ion(e.g. Tb³⁺).

As used herein, the term “binding member” refers to a portion of amarker molecule that is operably linked to a nucleotide molecule whereinsaid marker molecule further binds to another portion of a markermolecule so as to allow detection. For example, the present inventioncontemplates the detection of a photocleavable marker comprising a firstbinding member (e.g. biotin) that is detected by binding with a secondbinding member (e.g. streptavidin). In another example, the presentinvention contemplates the detection of a photocleavable markercomprising a first binding member (e.g. phenyldiboronic acid) that isdetected by binding with a second binding member (e.g. salicylhydroxamicacid).

As used herein, the term “BODIPY-FL” refers to a chemical compound(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid) that is fluorescent marker having the following chemicalstructure:

The term BODIPY-FL-SE refers to the succinimidyl ester of BODIPY-FL. Theterm BODIPY-FL-PC-aadUTP refers to BODIPY-FL that is operably linked to5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (aadUTP) via aphotocleavable linker.

As used herein, the term “Cy5” refers to a chemical compound that isfluorescent marker having the following chemical structure:

“Cy5” also refers to the chemical compound 1 [epsilon carboxypentyl]1′ethyl 3,3,3′,3′-tetramethylindocarbocyanine 5,5′-disulfonatepotassium salt N-hydroxysuccinamide ester.

As used herein, the term “photocleavable linker” refers to any chemicalgroup that attaches or operably links a (photocleavable) marker to thenucleobase moiety of a nucleotide, polynucleotide, or nucleic acid. Thepresent invention contemplates photocleavable linkers including, but notlimited to, 2-nitrobenzyl moieties, alpha-substituted 2-nitrobenzylmoieties [e.g. 1-(2-nitrophenyl)ethyl moieties], 3,5-dimethoxybenzylmoieties, thiohydroxamic acid, 7-nitroindoline moieties, 9-phenylxanthylmoieties, benzoin moieties, hydroxyphenacyl moieties, and NHS-ASAmoieties. The present invention also contemplates photocleavable linkerscomprising 2-nitrobenzyl moieties and “cross-linker arms” (or “spacerarms”) that further separate a photocleavable marker from the nucleobasemoiety of a nucleotide, polynucleotide, or nucleic acid to which it isto be operably linked. Examples of such “cross-linker arms” include, butare not limited to, long alkyl chains or repeat units of caproylmoieties linked via amide linkages.

As used herein, the term “protective group” refers to a chemical group(e.g. Fmoc and Bpoc) which is bound to a monomer unit and which may beselectively removed therefrom to expose an reactive or active site suchas, in the specific example of a nucleotide or photocleavable linker, anamine group. The present invention contemplates using protective groupsto enable (1) the sequential coupling of a photocleavable linker and amarker molecule, and (2) to prevent the reaction of an activatedphotocleavable linker with itself. The present invention contemplates,as depicted in FIG. 3 for example, that upon treatment of compound 4with ammonium hydroxide, the protective group is removed (e.g. compound5), thus allowing the directed interaction of the succinimidyl esterportion (i.e. the reactive site) of an active marker molecule with theexposed reactive site of a photocleavable linker to form aphotocleavable marker nucleotide or moiety (e.g. compounds 6 & 7).

As used herein, the term “reactive site” refers to the portion of amolecule or chemical group (or moiety) which is available to bind,operably link to, contact, or otherwise interact with another moleculeor chemical group after the removal of a protective group. The presentinvention contemplates photocleavable linkers that comprise a reactivesite upon the removal of a protective group such as Fmoc or Bpoc.

As used herein, the term monomer refers to a member of the set of smallmolecules which are or can be joined together to form a polymer. The setof monomers includes but is not restricted to, for example, the set ofnucleotides and the set of pentoses and hexoses. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.Furthermore, each of the sets may include protected members which aremodified after synthesis. The invention is described herein primarilywith regard to the preparation of molecules containing sequences ofmonomers such as nucleotides (including photocleavable markernucleotides), but could readily be applied in the preparation of otherpolymers. Such polymers include, for example, both linear and cyclicpolymers of nucleic acids.

As used herein, the term “polynucleic acid” refers to both linear andcyclic polymers of nucleic acids. An “unmodified polynucleic acid”refers to naturally occurring polynucleic acids. A “labeled polynucleicacid” refers to a polynucleic acid comprising a marker moiety.

As used herein, the term “template” refers to a nucleic acid moleculewhich may comprise single- or double-stranded DNA, RNA, or anoligonucleotide. The present invention contemplates the incorporation ofphotocleavable-marker nucleotides into such templates for variouspurposes including but not limited to nucleic acid sequencing.

As used herein, the term “nucleic acid-modifying enzyme” refers to anenzyme capable of modifying nucleic acids, nucleotides andpolynucleotides. Examples of such enzymes are well known in the art andinclude methylases (e.g. dam methylase), ligases (e.g. T4 DNA and RNAligase), nucleases (e.g. Exonuclease III and Mung Bean nuclease), andkinases (e.g. T4 Polynucleotide kinase and Uracil-DNA glycosylase). Theterm also refers to nucleic acid polymerase such as RNA polymerases(e.g. T7 and SP6 RNA polymerase) and DNA polymerases (e.g. Terminaldeoxynucleotidyl transferase, T4 and T7 DNA polymerase, thermophillicDNA polymerases, reverse transcriptases, DNA polymerase I, and DNApolymerase I Klenow fragment).

As used herein, the term “operably linked” refers to the linkage of achemical group or moiety (e.g. fluorophores, markers, and linkers) to anucleotide in such a manner that a bond that is capable of beingphotocleaved is produced. The term also refers to the linkage ofphosphate groups to a nucleotide in such a manner so that a nucleotidephosphate (e.g. nucleotide 5′ mono- or polyphosphate is produced. Ineither case, said nucleotide can be a single nucleotide, or apolynucleotide. The term “operably linking” refers to the act ofcreating an operably linked molecule, moiety or chemical group. Thepresent invention contemplates, for example, phosphate groups,photocleavable linkers and markers that are operably linked tonucleotides. The photocleavable agents of the present invention can be“operably linked” or “incorporated” into nucleotides and nucleic acids.By use of the term “operably linked” or “incorporated” it is not meantthat the entire photocleavable marker need be part of the finalmolecule. Some photocleavable agents of the present invention havereactive groups (i.e. the marker is an “activated marker”) and leavinggroups such that the photocleavable marker upon incorporation oroperable linkage may lose one or more groups.

As used herein, the term “sugar moiety” refers to the sugar molecule orgroup that is part of a nucleotide. For example, the present inventioncontemplates nucleotides comprising sugar moiety such as ribose anddeoxyribose. The present invention also contemplates “analogs” of saidsugar moieties such as dideoxyribose, and 2-fluoro-, 2-methoxy-, andacyclic sugar moiety analogs.

As used herein, the term “nucleobase” refers to a purine or pyrimidinebase attached to a 1′-carbon atom of a sugar moiety by an N-glycosidicbond to form a nucleoside. The present invention contemplates suchnucleobases as adenine, guanine, cytosine, thymine, and uracil, andanalogs thereof such as acyclic nucleobases and any of the known baseanalogs of DNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N-6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “nucleoside” refers to natural purinic andpyrimidinic nucleobases bound to sugar moieties. For example, thepresent invention contemplates nucleosides such as adenosine, cytidine,guanosine and uridine (i.e. for RNA) and deoxyadenosine, deoxycytidine,deoxyguanosine, and deoxythymidine (i.e. for DNA). The term “nucleosideanalogs” or refers to modified purinic and pyrimidinic nucleobases boundto sugar moieties such as 5-bromodeoxyuridine, deoxyinosine,deoxyuridine, 5-fluorodeoxyuridine, 5-iododeoxyuridine,5-methyldeoxycytidine, 3′-O-methylguanosine, 7-deaza-2′-deoxyadenosineand deoxyguanosine, and 2′-O-methyl-adenosine, cytidine, guanosine,inosine and uridine. Nucleosides that are bound to one (i.e. amonophosphate) or a plurality (i.e. a di-, tri- or polyphosphate) ofphosphate groups are referred to as “Nucleotides.” Examples ofnucleotides contemplated by the present invention include (but are notlimited to): 2′-deoxyuridine 5′-triphosphate (dUTP), 2′-deoxycytidine5′-triphosphate (dCTP), 2′-deoxyadenosine 5′-triphosphate (dATP),2′-deoxyguanosine 5′-triphosphate (dGTP), 2′-deoxyinosine5′-triphosphate (dITP), and 2′-deoxythymidine 5′-triphosphate (dTTP);and 2′,3′-dideoxyuridine 5′-triphosphate (ddUTP), 2′,3′dideoxyadenosine5′-triphosphate (ddATP), 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP),2′,3′dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′dideoxyinosine5′-triphosphate (ddITP), and 2′,3′dideoxythymidine 5′-triphosphate(ddTTP), and nucleotide analogs. The term “nucleotide analogs” refers tonucleotides which comprise various nucleoside analogs (e.g.5-fluorodeoxyuridine triphosphate, 5-iododeoxyuridine triphosphate,5-methyldeoxycytidine triphosphate, 3′-O-methylguanosine triphosphate,7-deaza-2′-deoxyadenosine and deoxyguanosine triphosphate, and2′-O-methyl-adenosine, cytidine, guanosine, inosine and uridinetriphosphate.

DESCRIPTION OF THE INVENTION

The present invention generally relates to nucleotides andpolynucleotides useful in the sequencing of nucleic acids. The presentinvention specifically relates to compositions comprising nucleotidesand polynucleotides comprising photocleavable markers. Such markers areuseful in DNA sequencing such as automated DNA sequencing employingfluorescent markers, various forms of parallel sequencing such assequencing by hybridization (see, e.g., Drmanac et al., (1998) NatureBiotechnol., 16, 54-58), pyrosequencing (see, e.g., Ronaghi et al.,(1998) Science, 281, 363-365) and in situ replica amplification. (See,e.g, R D Mitra and G M Church, “In situ localized amplification andcontact replication of many individual DNA molecules,” Nucl. Acids Res.,27(4): i-vi (1999); Published PCT Patent Application Nos. WO 99/19341and WO 00/53812 to Church & Mitra).

The compositions and methods of the present invention provide advantagesover those of the prior art it that the complex and time-consumingchemical methylation and enzymatic cleavage steps inherent in suchmethods are eliminated in favor of a rapid and simple photocleavagestep.

I. Compositions of the Present Invention

The present invention relates to compositions comprising nucleotides andpolynucleotides comprising photocleavable markers. Specifically, thepresent invention contemplates compositions comprising photocleavablemarker-polynucleotide conjugate compounds having the general formula(I), as depicted in FIG. 1, wherein X is selected from the groupconsisting of a phosphate group and a hydrogen atom, M is aphotocleavable marker, B is a nucleobase, PC-linker is a photocleavablelinker, and S is a sugar moiety.

It is not intended that the compounds of general formula (I) be limitedto a specific phosphate group. In one embodiment, said phosphate groupis a monophosphate group, more preferably a polyphosphate (such as adiphosphate group), and even more preferably a triphosphate group. Inanother embodiment, said phosphate group is a pyrophosphate group.

It is not intended that the nucleobase of the compounds of generalformula (I) be limited to a specific nucleobase. In one embodiment, saidnucleobase is selected from the group consisting of adenine, cytosine,guanine, thymine, uracil, and analogs thereof.

It is not intended that the sugar moiety of the compounds of generalformula (I) be limited to a specific sugar moiety. In one embodiment,said sugar moiety selected from the group consisting of ribose,deoxyribose, dideoxyribose, and analogs thereof, such as, for example,acyclic sugar moieties. (See, e.g., U.S. Pat. No. 5,558,991 to Trainoret al., “DNA Sequencing Method Using Acyclonucleoside Triphosphates”).

It is not intended that the photocleavable linker of the compounds ofgeneral formula (I) be limited to a specific photocleavable linker. Inone embodiment, said photocleavable linker is a photocleavable linkercomprising a protective group selected from the group consisting of9-fluorenylmethoxycarbonyl (Fmoc) and 2-(4-biphenyl)propyl(2)oxycarbonyl(Bpoc), and derivatives thereof (e.g. 9-fluorenylmethoxycarbonylN-hydroxysuccinimidyl ester; Fmoc-NHS).

It is not intended that the compounds of general formula (I) be limitedto any specific photocleavable marker. In one embodiment, saidphotocleavable marker is BODIPY-FL (FIG. 4) or its succinimidyl ester,BODIPY-FL-SE. In another embodiment, said photocleavable marker is Cy5,or its succinimidyl ester, Cy5-NHS. (FIG. 4). Succinimidyl esters arepreferred for the conjugation of dyes to nucleotides because they form avery stable amide bond between the dye and the nucleotide. The presentinvention also contemplates the use of other marker (or labels) such astetramethylrhodamine (6-TAMRA), fluorescein (5-FAM), rhodamine X(6-ROX), and 2′,7′-dimethoxy-4′,5′-dichlorofluorescein (6-JOE).Additional markers useful in conjunction with the present invention areshown in Table 1. For DNA sequencing applications, photocleavablemarkers comprising BODIPY moieties are useful because they areisomerically pure and cause little perturbation to the mobility of DNAfragments during polyacrylamide gel electrophoresis.

The present invention also contemplates compositions comprisingphotocleavable marker-polynucleotide conjugate compounds having thegeneral formula (II) (as depicted in FIG. 12), wherein X is selectedfrom the group consisting of a phosphate group and a hydrogen atom, M isa photocleavable marker, B is a nucleobase, and S is a sugar moiety.

It is not intended that the compounds of general formula (II) be limitedto a specific phosphate group. In one embodiment, said phosphate groupis a monophosphate group, more preferably a polyphosphate (such as adiphosphate group), and even more preferably a triphosphate group. Inanother embodiment, said phosphate group is a pyrophosphate group.

It is not intended that the nucleobase of the compounds of generalformula (II) be limited to a specific nucleobase. In one embodiment,said nucleobase is selected from the group consisting of adenine,cytosine, guanine, thymine, uracil, and analogs thereof.

It is not intended that the sugar moiety of the compounds of generalformula (II) be limited to a specific sugar moiety. In one embodiment,said sugar moiety selected from the group consisting of ribose,deoxyribose, dideoxyribose, and analogs thereof, such as, for example,acyclic sugar moieties. (See, e.g., U.S. Pat. No. 5,558,991 to Trainoret al., “DNA Sequencing Method Using Acyclonucleoside Triphosphates”).

It is not intended that the photocleavable linker of the compounds ofgeneral formula (II) be limited to a specific photocleavable linker. Inone embodiment, said photocleavable linker is a photocleavable linkercomprising a protective group selected from the group consisting of9-fluorenylmethoxycarbonyl (Fmoc) and 2-(4-biphenyl)propyl(2)oxycarbonyl(Bpoc), and derivatives thereof (e.g. 9-fluorenylmethoxycarbonylN-hydroxysuccinimidyl ester; Fmoc-NHS).

It is not intended that the compounds of general formula (II) be limitedto any specific photocleavable marker. In one embodiment, saidphotocleavable marker is BODIPY-FL (FIG. 4) or its succinimidyl ester,BODIPY-FL-SE. In another embodiment, said photocleavable marker is Cy5,or its succinimidyl ester, Cy5-NHS. (FIG. 4). Succinimidyl esters arepreferred for the conjugation of dyes to nucleotides because they form avery stable amide bond between the dye and the nucleotide. The presentinvention also contemplates the use of other marker (or labels) such astetramethylrhodamine (6-TAMRA), fluorescein (5-FAM), rhodamine X(6-ROX), and 2′,7′-dimethoxy-4′,5′-dichlorofluorescein (6-JOE). For DNAsequencing applications, photocleavable markers comprising BODIPYmoieties are useful because they are isomerically pure and cause littleperturbation to the mobility of DNA fragments during polyacrylamide gelelectrophoresis. Additional markers useful in conjunction with thepresent invention are shown in Table 1.

One example of the photocleavable markers found in the compounds ofgeneral formulas (I) & (II) contemplated by the present invention arechemical compounds which contain, or are operably linked to, a2-nitrobenzyl moiety. (See, e.g., U.S. Pat. Nos. 5,922,858 & 5,643,722to Rothschild et al.). Upon illumination, these aromatic nitro compoundsundergo an internal oxidation-reduction reaction (V. N. RajasekharanPillai, “Photoremovable Protecting Groups in Organic Synthesis,”Synthesis, 1:1-26 (1980); Patchornik et al., (1970) J. Am. Chem. Soc.92: 6333-35). As a result, the nitro group is reduced to a nitroso groupand an oxygen is inserted into the benzylic carbon-hydrogen bond at theortho position. The primary photochemical process is the intramolecularhydrogen abstraction by the excited nitro group. This is followed by anelectron-redistribution process to the aci-nitro form which rearrangesto the nitroso product. Subsequent thermal reaction leads to thecleavage of substrate from the nitrobenzyl linkage. Examples ofphotocleavable markers of the present invention are shown in FIG. 4.

TABLE 1 Name and Molecular weight Formula Fluorescence PropertiesBODIPY-FL, SSE M. WT. 491

Excitation = 502 nm Emmision = 510 nm Extinction = 75,000 NBD M. WT. 391

Excitation = 466 nm Emmision = 535 nm Extinction = 22,000 Bodipy-TMR-X,SE M. WT. 608

Excitation = 544 nm Emmision = 570 nm Extinction = 56,000 Bodipy-R6G M.WT. 437

Excitation = 528 nm Emmision = 547 nm Extinction = 70,000 Fluorescein(FAM) M. WT. 473

Excitation = 495 nm Emmision = 520 nm Extinction = 74,000 Fluorescein(SFX) M. WT. 587

Excitation = 494 nm Emmision = 520 nm Extinction = 73,000 PyMPO M. WT.582

Excitation = 415 nm Emmision = 570 nm Extinction = 26,000 5/6-TAMRA M.WT. 528

Excitation = 546 nm Emmision = 576 nm Extinction = 95,000

In another embodiment, the compounds of general formulas (I) & (II) arechemical compounds which contain, or are operably linked to aphotocleavable linker selected from the group consisting ofalpha-substituted 2-nitrobenzyl moieties [e.g. 1-(2-nitrophenyl)ethylmoieties], 3,5-dimethoxybenzyl moieties, thiohydroxamic acid,7-nitroindoline moieties, 9-phenylxanthyl moieties, benzoin moieties,hydroxyphenacyl moieties, and NHS-ASA moieties.

It may sometimes be desirable to create a distance between the substrate(e.g. such as nucleotides, polynucleotides, oligonucleotides, or nucleicacids) and the photocleavable marker moiety. To accomplish this,photocleavable moieties may be separated from substrates by cross-linkerarms. Cross-linkers increase substrate access and stabilize the chemicalstructure, and can be constructed using, for example, long alkyl chainsor multiple repeat units of caproyl moieties linked via amide linkages.

In one embodiment, the marker BODIPY-FL is operably linked to a an alkylchain cross-linker via the marker's succinimidyl ester group, and saidcross-linker is directly attached to a 2-nitrobenzyl moiety linked to anucleotide moiety. (See FIG. 3, synthesis of compound 6 from compound5). In another embodiment, the marker Cy5 is operably linked asdescribed above. (See FIG. 3, synthesis of compound 7 from compound 5).Other examples of photocleavable markers include photocleavablecoumarin, photocleavable dansyl, photocleavable dinitrophenyl andphotocleavable coumarin-biotin.

Photocleavable markers are cleaved by electromagnetic radiation such asUV light. Cleavage of photocleavable markers is dependent on thestructure of the photoreactive moiety and the wavelength ofelectromagnetic radiation used for illumination. Other wavelengths ofelectromagnetic radiation should not damage nucleotides or otherchemical moieties to which the photocleavable marker is bound, attachedor operably linked. Typical illumination times vary from less than 1hour (e.g. 1 minute to thirty minutes) to about 24 hours and radiationor illumination sources are placed within about 10 cm of the reactionmixture (and set on low power so as to minimize side reactions, if any,which may occur).

It is not intended that the photocleavable marker of the compounds ofgeneral formulas (I) & (II) be detected by any specific method. In oneembodiment, said photocleavable marker is a molecule that can bedetected by mass spectrometry. In another embodiment, saidphotocleavable marker is a fluorescent moiety and can be detected byfluorescence spectroscopy.

In another embodiment, said photocleavable marker is a binding memberand is detected via a second binding member. Specifically, the presentinvention contemplates photocleavable markers wherein a portion of amarker molecule is operably linked to a nucleotide molecule. Said markermolecule is further bound to another portion of a marker molecule so asto allow detection. For example, in one embodiment, the presentinvention contemplates the detection of a photocleavable markercomprising biotin as a first binding member, that is detected by bindingwith streptavidin as the second binding member. In another embodiment,said first binding member is phenyldiboronic acid and salicylhydroxamicacid is said second binding member.

In a preferred embodiment, said photocleavable marker is a chelatorcapable of forming luminescent complexes. In principle, a first chelatoris incorporated into a nucleic acid by being operably linked to aphotocleavable nucleotide. A second chelator is added free in solutionwith a metal ion and the luminescent complex is formed. Examples of someof the first and second chelators, and metal ions, contemplated by thepresent invention are summarized in Table 2 below. The present inventioncontemplates embodiments wherein said first and second chelators are thesame molecule, as well as embodiments in which said first and secondchelators are different molecules. (See, e.g., Table 2).

In one embodiment, a first chelator is incorporated into immobilizedpolynucleotide, followed by the addition of a second chelator and metalion such that a luminescent complex is formed. Excess (i.e. unbound)second chelator and metal ion are washed away, and said complex isdetected by luminescence assay. In another embodiment, said complex isdetected in solution by performing a dissociation wherein an excess of acompeting chelator (e.g. “BCPDA” or4,7-bis(chlorosulfophenyl)-1,10-phenantroline-2,9-dicarboxylic acid) andan enhancing agent (such as, for example, a detergent) are added to thefirst chelator incorporated into an immobilized polynucleotide. (See I.Hemmila, “Applications of Fluorescence in Immunoassays,”Wiley-Interscience, New York, 1991).

TABLE 2 First Chelator Second Chelator Metal Ion(s) salicylic acid EDTATb³⁺ 3-hydroxypyridine 1,10-phenantroline Eu³⁺ β-diketone (β-naphthoyl-EDTA Tb³⁺, Eu³⁺, trifluoroacetone) or Sm³⁺ β-diketone (β-pivaloyl- EDTATb³⁺ trifluoroacetone) or Eu³⁺ 2,2′-bipyridine EDTA or 2,2′-bipyridineRu³⁺

II. Methods of the Present Invention

A. Photocleavable Marker-Polynucleotide Conjugates

The present invention further relates to the methods of preparingphotocleavable marker-polynucleotide conjugates. As depicted in FIG. 2,the overall method of the present invention involves the incorporationof photocleavable marker-nucleotide conjugates into polynucleotides,nucleic acids, polynucleic acids, and other suitable templates. Onceincorporated into a polynucleotide or polynucleic acid, thephotocleavable marker is detected by such methods as luminescence,fluorescence, chemiluminescence or mass spectrometry. After detection ofthe photocleavable marker, said marker is removed by photocleavage (e.g.by UV irradiation) and washed away to separate free (i.e. cleaved)marker-nucleotides from the reaction mixture. The entire process is thenrepeated again with the same photocleavable marker-nucleotide beingincorporated into a different position on the same nucleic acid orpolynucleic acid, followed by detection of the photocleavable marker,etc. as described above. However, it important to note that the presentinvention also contemplates embodiments in which differentphotocleavable marker-nucleotide and nucleotide conjugates are employedin the subsequent incorporation steps of the above process. Such anembodiment employs two or more different marker moieties that can beindependently detected (i.e. each marker has a distinct UV-VISabsorbance spectra such that they are distinguishable upon signaldetection as contemplated herein). For example, FIG. 8 depicts acomparison of the UV-VIS absorbance spectra for BODIPY-FL dye (˜270 nm)and the photocleavable marker-nucleotide, BODIPY-FL-PC-aadUTP (compound6)(˜370 nm). Moreover, FIG. 9 depicts a comparison of the U-VISabsorbance spectra for Cy5 dye (˜270 nm) and the photocleavablemarker-nucleotide, Cy5-PC-aadUTP (compound 7)(˜650 nm). When takentogether, the UV-VIS spectral values indicated in FIGS. 8 and 9 indicatethat a method of incorporating a photocleavable marker-nucleotideconjugate into the same polynucleic acid would allow the independentdetection of said markers since both photocleavable marker-nucleotideare distinguishable over the fluorophore dyes and starting material(aadUTP) of which they are comprised, as well as, each other.

It is not intended that the present invention be limited to a specificmethod of incorporating photocleavable marker-nucleotides intopolynucleotides, nucleic acids, polynucleic acids, oligonucleotides, andother suitable templates (to form photocleavable marker-polynucleotideconjugates). In one embodiment, said incorporation involves theenzymatic incorporation of the photocleavable marker-nucleotideconjugate BODIPY-FL-PC-aadUTP into an oligonucleotide specific for thehuman Cystic Fibrosis Transmembrane Regulator Gene using the GeneImages3′ oligolabelling kit (AP-Biotech) (as per the manufacturersinstructions) wherein an unmodified polynucleic acid or polynucleotideis incubated with said marker-nucleotide 5′ triphosphate conjugate and aDNA or RNA modifying enzyme such as terminal deoxynucleotidyltransferase. In another embodiment, the photocleavable marker-nucleotideconjugate Cy5-PC-aadUTP is incorporated. FIG. 10 depicts an example ofsuch an incorporation of BODIPY-FL-PC-aadUTP into a polynucleic acid orpolynucleotide.

It is not intended that the present invention be limited to a specificmethod of detecting a photocleavable marker-nucleotide conjugate. In oneembodiment, said method of detecting is selected from the groupconsisting of luminescence, fluorescence, chemiluminescence or massspectrometry. For example, in one embodiment, said photocleavable markeris detected by denaturing polyacrylamide gel electrophoresis followed byfluorescence image scanning before photocleavage has occurred. (See,e.g., FIG. 10). Note that such detection of photocleavable markers maybe accomplished before or after photocleavage of the photocleavablemarker-nucleotide (or photocleavable marker-polynucleotide conjugate).(See, e.g., FIG. 11).

It is not intended that the present invention be limited to a particularmeans by which a photocleavable marker is cleaved. For example, in oneembodiment, a photocleavable marker comprising BODIPY-FL is cleaved fromits nucleotide conjugate after being subjected to irradiation by near UVlight (300-365 nm, ˜1 mW/cm²) for five minutes. In another embodiment, aphotocleavable marker comprising Cy5 is cleaved from its nucleotideconjugate under the same conditions (and in the same manner) asdescribed above. FIG. 6 depicts HPLC chromatograms of photocleavablemarker nucleotides of the present invention comprising either BODIPY-FL(compound 6) or Cy5 (compound 7) before and after UV irradiation asdescribed above. FIG. 6 indicates that UV irradiation cleaves thephotocleavable moiety from the nucleotide to which it was operablylinked with the conversion of compounds 6 & 7 to compound 3 (aadUTP)(i.e. fluorophore removal was successful).

B. Photocleavable Marker-Nucleotide Conjugates

The present invention also relates to the methods of preparingphotocleavable marker-nucleotide conjugates. It is not intended that thepresent invention be limited to a particular method of preparingphotocleavable marker-nucleotide conjugates. In one embodiment, a methodfor the synthesis of a photocleavable marker-nucleotide conjugatecomprising a fluorophore selected from the group consisting of BODIPY-FL(i.e. resulting in compound 6) or Cy5 (i.e. resulting in compound 7) isas depicted by the chemical synthesis scheme of FIG. 3.

Briefly, compound 1 comprising the protective group, Fmoc, was preparedas described in Olejnik et al., (1998), Methods Enzymol., 291: 135-54,and reacted in acetonitrile, with N,N-diisopropylethylamine (DIPEA) andN,N′-disuccinimidyl carbonate (DSC) under conditions such that theintermediate compound 2 was formed. Compound 2 purified bychromatography and reacted with an aminoallyl-deoxynucleotidetriphosphate (e.g. aadUTP) under conditions such that compound 4 wasformed. Compound 4 was purified by reverse phase high performance liquidchromatography (RP-HPLC), and subsequently reacted with ammonia suchthat the Fmoc protective group was removed and compound 5 was produced.Compound 5 was also purified by RP-HPLC and then incubated with thesuccinimidyl ester of a fluorophore selected from BODIPY-FL (to makecompound 6) or Cy5 (to make compound 7). Compounds 6 & 7 were analyzedby photocleavage and HPLC. (See, e.g., FIGS. 6 & 8).

DESCRIPTION OF PREFERRED EMBODIMENTS

As noted above, the compositions of the present invention are useful inDNA sequencing such as automated DNA sequencing employing fluorescentmarkers, various forms of parallel sequencing such as sequencing byhybridization. For example, the present invention contemplates theutilization of photocleavable marker-nucleotides in the method to cloneand amplify DNA by PCR as taught in R D Mitra and G M Church, “In situlocalized amplification and contact replication of many individual DNAmolecules,” Nucl. Acids Res., 27(4): i-vi (1999), herein incorporated byreference. In Mitra & Church, a method to clone and amplify DNA byperforming PCR in a thin polyacrylamide film poured on a glassmicroscope slide. Id. The polyacrylamide matrix retards the diffusion ofthe linear DNA molecules so that the amplification products remainlocalized near their respective templates. Id. At the end of thereaction, a number of PCR colonies have formed, each one “grown” from asingle template molecule, with as many as five million clones amplifiedand sequenced in parallel on a single slide using asequencing-by-synthesis method such as pyrosequencing. Id. This isusually adequate for gene identification or mini-sequencing. However, anew sequencing-by-synthesis method, fluorescent in situ sequencingextension quantitation (FISSEQ), is particularly suitable. Id.

Briefly, in FISSEQ, the DNA is extended by adding a single type offluorescently-labeled nucleotide triphosphate to the reaction, followedby the washing away of unincorporated nucleotide, detecting theincorporation of the nucleotide by measuring fluorescence, and repeatingthe cycle until synchrony is lost. At each cycle, the fluorescence fromprevious cycles is “bleached” or digitally subtracted, allowing one todeduce the sequence of each polony iteratively. In a preferredembodiment, the present invention contemplates the utilization of thephotocleavable marker-nucleotides described herein as a source offluorescently-labeled nucleotide triphosphates in the FISSEQ method.Said photocleavable marker-nucleotides provide the added advantage overthe method of Mitra & Church by allowing a simplified, expedient,non-enzymatic cleavage (i.e. the cleavage of the fluorescent marker ofthe present invention is by photolysis) of the fluorescent marker moietyfrom the nucleic acid (or polynucleic acid) into which it wasincorporated.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); FITC (fluoresceinisothiocyanate); M (Molar); μM (micromolar); N (Normal); mol (moles);mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanogram); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); rpm(revolutions per minute); EDTA (ethylenediaminetetracetic acid); dCTP(2′-deoxycytidine 5′-triphosphate); dUTP (2′-deoxyuridine5′-triphosphate); Roche Molecular (Roche Molecular Biochemicals,Indianapolis, Ind.); Gibco-BRL (Gibco-BRL Life Technologies, Inc.,Rockville, Md.); Molecular Probes (Molecular Probes, Eugene, Oreg.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Promega (Promega Corp.,Madison, Wis.); AB (Applied Biosystems, Foster City, Calif.).

Example 1 Synthesis of Photocleavable BODIPY-FL DeoxyuridineTriphosphate (BODIPY-FL-PC-aadUTP) (FIGS. 3, 4)

In this example, one method for the production of the photocleavablemarker-nucleotide conjugate, BODIPY-FL-PC-aadUTP (compound 6), isdescribed.

A. Synthesis of Intermediate Compounds (Compounds 2, & 5)

Compound 1 (Olejnik, J., E. Krzymanska-Olejnik, and K. J. Rothschild.1998. Methods Enzymol. 291:135-54) (100 mg, 0.19 mmol) was dissolved inanhydrous acetonitrile (10 ml) and to this solution 50 μl (0.285 mmol,1.5 eq.) of N,N-diisopropylethylamine (DIPEA) (Sigma Cat. No. D 3887)was added followed by N,N′-disuccinimidyl carbonate (DSC) (Sigma Cat.No. D 3773) (75 mg, 0.285 mmol, 1.5 eq.). The mixture was stirred atroom temperature overnight, volatile compounds removed under reducedpressure and the intermediate (compound 2) purified on a silica gelcolumn using a step (0-1.5%) gradient of MeOH in CHCl₃ with a yield of500 mg (39%).

To make compound 5, 1 mg (1.9 μmol) of 5-(3-aminoallyl)-2-deoxyuridine5′-triphosphate (compound 3) (aadUTP) (Sigma Cat. No. A 5660) wasdissolved in 100 μl of 50 mM NaHCO₃ (pH 8.5). To this solution, asolution of 5 mg of compound 2 (7.6 μmol, 4 eq.) in 200 μl ofacetonitrile was added. The mixture was incubated at room temperaturefor 2 hours and purified using preparative RP-HPLC (Waters NovaPak C18,10×100 mm) using 0-90% gradient of acetonitrile in 50 mMtriethylammonium acetate (pH 4.5) over a period of 45 minutes with flowrate 1 ml/min. The fractions containing compound 4 were pooled andfreeze dried to give ˜1 μmol of material. This material was dissolved in1 ml of water, and to this solution, 200 μl of concentrated ammonia wasadded. The solution was incubated overnight at room temperature,freeze-dried and compound 5 purified using RP-HPLC as described abovewith a yield of 0.6 μmol.

B. Synthesis of BODIPY-FL-PC-aadUTP from Intermediate Compounds

Compound 5 (0.23 μmol) was dissolved in 100 μl of 50 mM NaHCO₃ and then73 μl of a 25 mM solution of BODIPY-FL-SE in dimethylformamide (DMF)(Molecular Probes Cat. No. D-2184) was added. The reaction mixture wasincubated for two hours at room temperature and the product isolatedusing RP-HPLC as described above. Fractions containing the desiredproduct were pooled and freeze-dried to give 36 nmoles of compound 6(based on BODIPY-FL fluorophore absorption, Absorption max=505 nm,ε=80,000).

Compound 6 was further characterized by photocleavage and HPLC analysisas well as absorption spectra extracted from the HPLC traces. For eachof these experiments, approximately 2 mmoles of the material (i.e.compound 6) was used. The results of these experiments are depicted inFIGS. 6 and 8.

Example 2 Synthesis of Photocleavable Cy5 deoxyuridine triphosphate(Cy5-PC-aadUTP)

In this example, one method for the production of the photocleavablemarker-nucleotide conjugate, Cy5-PC-aadUTP (compound 7), is described.

Compound 5 (0.24 μmol), as prepared above, was dissolved in 40 μl of 50mM NaHCO₃, followed by the addition of 0.72 μmol of a Cy5-NHS(Amersham-Pharmacia Biotech Cat. No. PA 25001) solution in 100 μl ofDMF. The reaction mixture was incubated for 2 hours at room temperatureand the product was isolated using RP-HPLC initially on R2/10 RP column(Perseptive Biosystems, 4.6×100 mm) followed by another purification onNovaPak C18, (Waters, 10×100 mm). In both case a gradient (0-90%) ofacetonitrile in 50 mM triethylammonium acetate (pH 4.5) over 45 minuteswith flow rate 1 ml/min. was used. Fractions containing the desiredproduct were pooled and freeze-dried to give 60.5 nmoles of compound 7(based on Cy5 fluorophore 550 nm absorption maximum, ε=250,000).

Compound 7 was further characterized by photocleavage and HPLC analysisas well as absorption spectra extracted from the HPLC traces. For eachof these experiments, approximately 2 nmoles of the material (i.e.compound 7) was used. The results of these experiments are depicted inFIGS. 6 and 8.

Example 3 Enzymatic Incorporation of BODIPY-FL-PC-aadUTP into DNA andPhotocleavage

In this example, one method for the incorporation of photocleavablemarker-nucleotide into a nucleic acid (or polynucleic acid) to form aphotocleavable marker-polynucleotide conjugate is described. Althoughthe example below specifies the usage of BODIPY-FL-PC-aadUTP, it isimportant to note that the present invention also contemplates a methodfor the incorporation of photocleavable marker-nucleotide into a nucleicacid (or polynucleic acid) to form a photocleavablemarker-polynucleotide conjugate wherein Cy5-PC-aadUTP is substituted inplace of BODIPY-FL-PC-aadUTP (i.e. the method below will work equallywell with either photocleavable marker nucleotide).

The enzymatic incorporation of a photocleavable marker-nucleotide intothe oligonucleotide was performed using components of commerciallyavailable kit (Amersham-Pharmacia Biotech, Gene Images 3′-oligolabelingkit, Cat. No. RPN 5770) per the manufacturers instructions.

Briefly, an oligodeoxynucleotide (30-mer) having the sequence:5′-GTA-TCT-ATA-TTC-ATC-ATA-GGA-AAC-ACC-ACA-3′ (SEQ ID NO: 1) was used.This primer is useful for the amplification of a fragment of the humanCFTR (Cystic Fibrosis Transmembrane Regulator) gene.

A volume of 10 μl of oligodeoxynucleotide (25 pmoles), water (5.8 μl),BODIPY-FL-PC-aadUTP (0.38 nmol, 1.25 μl), cacodylate buffer (2 μl) andterminal deoxynucleotidyl transferase (TdT) were mixed in a 500 μlmicrocentrifuge tube and incubated at 37° C. for 1 hour. An aliquot (1μl) of the mixture was loaded on a denaturing 7M urea/15% polyacrylamidegel and imaged using a fluorescence scanning device (FluorImager,Molecular Dynamics). A control experiment utilizing Fluorescein-11-dUTPwas also performed and analyzed on the same gel. The results of theseexperiments are shown in FIG. 10. Both in the control experiment(Fluorescein-11-dUTP) and in the experiment utilizingBODIPY-FL-PC-aadUTP, a generation of several fluorescent bands wereobserved. These are most likely due to addition of multiple labels onthe 3′-end by terminal transferase.

In a separate experiment an aliquot of BODIPY-FL-PC-aadUTP labeled DNAwas subjected to near UV irradiation (300-365 nm, ˜1 mW/cm²)(BlakRayXX-15, UVP, Inc., San Gabriel, Calif.) for 5 minutes prior to gelanalysis and imaging. The results of this experiment are depicted inFIG. 11. The fluorescent signal observed in BODIPY-FL-PC-aadUTP labeledoligonucleotide disappears completely after UV irradiation, whichindicates that the fluorescent label has been removed (i.e. cleaved off)during UV irradiation.

1. A conjugate, comprising a nucleotide attached to a fluorescent markerthrough a cleavable linker, said marker comprising BODIPY.
 2. Theconjugate of claim 1, wherein said nucleotide is a ribonucleotide. 3.The conjugate of claim 1, wherein said nucleotide is adeoxyribonucleotide.
 4. The conjugate of claim 1, wherein said cleavablelinker is a photocleavable linker.
 5. A method, comprising: a) providingi) nucleic acid, ii) the conjugate of claim 1, and iii) a nucleicacid-modifying enzyme; b) mixing said nucleic acid and said nucleicacid-modifying enzyme in the presence of said conjugate under conditionssuch that said conjugate is incorporated into said nucleic acid toproduce labeled nucleic acid.
 6. The method of claim 5, wherein saidnucleic acid is RNA.
 7. The method of claim 5, wherein said nucleic acidis DNA.
 8. The method of claim 5, wherein said nucleic acid-modifyingenzyme is a polymerase.
 9. The method of claim 5, wherein said nucleicacid-modifying enzyme is a terminal transferase.
 10. The method of claim5, wherein said nucleic acid-modifying enzyme is a ligase.